An extensive literature, accumulated over the past forty years, demonstrates that variation of essentially any aspect of membrane lipid composition leads to modulation of the functional efficacy and/or oligomeric state of one or more membrane proteins. The effects that membrane composition may also have on membrane protein folding and structural stability have received somewhat less attention. Characterizing the fundamental forces that determine the thermodynamic stability of membrane proteins is an area of intense interest. The energetic contribution of lipid-protein interactions to membrane protein stability is likely to be as important for this class of proteins as the energetics of protein-solvent interactions is to the stability of soluble proteins. Determining the effects of membrane composition on the energy required for thermal denaturation provides a measure of the effects of membrane composition on relative protein folding energetics.[unreadable] Purified bovine rhodopsin was reconstituted into vesicles containing a range of cholesterol and docosahexaenoic acid (DHA, 22:6n3) acyl chains and rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate dependent, demonstrating the presence of a kinetically controlled component of denaturation. The activation energy of this kinetically controlled process (Ea) was determined from DSC thermograms by four separate methods. Both Tm and Ea varied with bilayer composition. The thermal stability of rhodopsin in SOPC reconstituted membranes was increased by the addition of 30 mol% cholesterol, and decreased by the addition of 20% SDPC. The simultaneous addition of cholesterol and SDPC further enhances the thermostability of rhodopsin beyond the effects of cholesterol alone, which may suggest the promotion of rhodopsin-cholesterol interactions by SDPC. Cholesterol increased Tm regardless of the level of DHA acyl chains. In contrast, cholesterol lowered Ea in the absence DHA acyl chains, but raised Ea in the presence of 20 mol% DHA-containing phospholipid. Relative acyl chain packing order was determined from measurements of diphenylhexatriene fluorescence anisotropy decay. Tm for thermal unfolding decreased with increasing acyl chain disorder while kinetic stability (Ea) was maximal over a middle range of acyl chain order, such that highly ordered or disordered membranes resulted in kinetic destabilization. The activation energies of thermal denaturation in each of the samples, when considered as a function of acyl chain packing, demonstrate that the kinetic component of rhodopsin denaturation is maximally stabilized in membranes whose lipid packing is similar to that of the native disk membrane. These results establish that one important function of the combination of DHA acyl chains and cholesterol found in the rod outer segment disk membrane is to provide maximal kinetic stability for rhodopsin. The results also demonstrate that membrane composition has distinct effects on the thermal and kinetic stabilities of membrane proteins and suggest that a balance between membrane constituents that have opposite effects on acyl chain packing, such as DHA and cholesterol may be required for maximum protein stability.